Mechanical holder for surface analysis

ABSTRACT

A mechanical holder that provides for a confined sampling region for extraction and removal of chemical substances contained in a dried blood spot or other spot of sample is described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 61/333,956, filed on May 12, 2010,which is incorporated by referenced herein in its entirety.

BACKGROUND

Mass spectrometry is an analytical process that identifies the chemicalcomposition of a compound or sample based on the mass-to-charge ratio ofcharged particles derived from the compound or sample. In general, inmass spectrometry, a sample undergoes ionization to form chargedparticles (ions). The ratio of mass-to-charge of the particles isdetermined by passing them through electric and/or magnetic fields in amass spectrometer.

In some mass spectrometer systems, molecules can be analyzed in aquadrupole mass spectrometer using “electrospray” ionization tointroduce the ions into the spectrometer. In electrospray ionization aspray needle may be positioned near to the entrance orifice of aquadrupole, magnetic, ion trap, Fourier transform mass spectrometer(FTMS), orbitrap, or time-of-flight (TOF) mass spectrometer, or close tothe entrance of a capillary leading to a vacuum entrance orifice of thequadrupole or other type of mass spectrometer. A dilute solution,including the molecules of interest, is pumped through the electrosprayneedle or emitter and an electric potential between the needle oremitter orifice and a vacuum orifice (e.g., a reference electrode)leading to the mass analyzer forms a spray (“electrospray”) of thesolution.

SUMMARY

Dried blood spots (DBS) or dried matrix spots (DXS) where X can be anysample type on an sample card such as paper or other substrate surfacesare viewed as an alternative to the conventional methods of collecting,transporting, and storing biological samples such as urine, plasma,saliva and feces as well as milk and other samples of interest. Amechanical holder that provides for a confined sampling region forextraction and removal of chemical substances contained in a driedmatrix spot with direct analysis of the extracted sample is describedherein. Methods for analyzing a dried matrix spots such as dried bloodspots are also described herein.

In some aspects, a method of extracting analytes from a sample cardincludes placing a sample card that includes a dried sample in a lowerportion of a device configured to house the sample card and lowering anupper portion of the device mechanically connected to the lower portion,the upper portion including one or more fittings with each fittingdefining an opening in the upper portion and including a rigid loweredge. The method also includes engaging one or more locking mechanismsto compress the sample card substantially uniformly between the rigidlower edge of the one or more fittings and the lower portion to form asubstantially liquid tight seal around a portion of the sample card. Themethod also includes introducing a solvent into a solvent deliverydevice, dispensing the solvent to contact a surface of the sample card,and aspirating the solvent and dissolved analytes into the solventdelivery device from the surface of the sample card. The method alsoincludes interfacing the solvent delivery device to an automation deviceto deliver the solvent delivery device to an ion sprayer and directingions from the ion sprayer to a mass analyzer.

Embodiments can include one or more of the following:

The sample card can be a dried blood spot card.

The method can also include repeating the steps of dispensing andaspirating prior to interfacing the solvent delivery device to anautomation device to deliver the solvent delivery device to the ionsprayer.

The method can also include repeating the steps of dispensing andaspirating at the same location on the sample prior to interfacing thesolvent delivery device to an automation device to deliver the solventdelivery device to the ion sprayer.

Dispensing the solvent can include dispensing microliters of solventfrom the first end of the solvent delivery device to contact the sample.

Engaging one or more locking mechanisms can depress the sample card bybetween about 0.015 inch to about 0.030 inch to form the substantiallyliquid tight seal.

Engaging one or more locking mechanisms can depress the sample card bybetween about 0.010 in to 0.100 in to form the substantially liquidtight seal.

Engaging one or more locking mechanisms can depress the sample card bybetween about 0.025 inch to about 0.1 inch to form the substantiallyliquid tight seal.

Engaging one or more locking mechanisms can include tightening one ormore screws.

Dispensing the solvent can include forming a liquid junction between asample surface of the sample card and the solvent delivery device.

Dispensing the solvent can include wetting a surface of the sample card.

Engaging the one or more locking mechanisms can include engagingmultiple locking mechanisms to apply a substantially uniform pressureonto sample substrate surface by the upper portion.

In some additional aspects, a system can include a lower portionconfigured to house a sample card that includes a dried sample, an upperportion mechanically connected to the lower portion, the upper portionincluding one or more fittings each defining an opening in the upperportion configured to receive a solvent delivery device and including arigid lower perimeter edge, and one or more locking mechanismsconfigured to compress the sample card between the rigid lower perimeteredge of the one or more fittings and the lower portion to form asubstantially liquid-tight seal around a portion of the sample card whenthe upper portion and the lower portion are in a closed position and thelocking mechanisms are engaged.

Embodiments can include one or more of the following:

The solvent delivery device can be capable of delivering an extractionsolvent for extracting chemicals from the sample substrate material.

The solvent delivery device can be a pipette tip.

The solvent delivery device can be an extraction tip.

The fitting can be a PEEK, polymer, brass or stainless steel with arigid lower circular perimeter edge capable of making a leak-tight sealwhen clamped down upon the sample card without cutting a disk from thesample card.

The upper portion can include at least four fittings in an array.

The upper portion can include 96 fittings in an array.

The upper portion can include between four and 96 fittings in an array.

The system can also include a robotic device configured to extractanalytes from the substrate to form a solution from a surface of thesample card using a solvent delivery device which can then be withdrawnfrom the surface and delivered to a mass spectrometer.

The fitting can be configured such that the extraction solventintroduced into the fitting is substantially confined within the wallsof the fitting.

Confining the extraction solvent within the walls of the fitting canpreclude the need to employ hydrophobic spray chemicals to preclude saiddispersion.

The liquid-tight seal can be configured to confine at least 90% of theextraction solvent within the walls of the fitting.

The liquid tight seal can be configured to confine at least 95% of theextraction solvent within the walls of the fitting.

The liquid tight seal can be configured to confine at least 98% of theextraction solvent within the walls of the fitting.

In some additional aspects, a system includes a lower portion configuredto house a sample card and an upper portion mechanically connected tothe lower portion, the upper portion including a fitting or an array offittings configured such that when the upper portion and the lowerportion are in a closed position, a substantially liquid-tight seal isformed around a portion of the sample card.

Embodiments can include one or more of the following:

The solvent delivery device can be capable of delivering an extractionsolvent for extracting chemicals from the sample substrate material.

The solvent delivery device can be a pipette tip.

The solvent delivery device can be an extraction tip.

The fitting can be a PEEK, polymer, brass or stainless steel with arigid lower circular perimeter edge capable of making a leak-tight sealwhen clamped down upon the sample card without cutting a disk from thesample card.

The upper portion can include at least four fittings in an array.

The system can also include a robotic device configured to extractanalytes from the substrate to form a solution from a surface of thesample card using a solvent delivery device which can then be withdrawnfrom the surface and delivered to a mass spectrometer.

The fitting can be configured such that extraction solvent introducedinto the fitting is substantially confined within the walls of thefitting.

Confining the extraction solvent within the walls of the fitting canpreclude the need to employ hydrophobic spray chemicals to preclude saiddispersion.

The liquid tight seal can be configured to confine at least 75% of theextraction solvent within the walls of the fitting.

The liquid tight seal can be configured to confine at least 90% of theextraction solvent within the walls of the fitting.

The liquid tight seal can be configured to confine at least 95% of theextraction solvent within the walls of the fitting.

The liquid tight seal can be configured to confine at least 98% of theextraction solvent within the walls of the fitting.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C show an exemplary mechanical holder system.

FIGS. 2A and 2B show an exemplary pipette inserted into a fitting in themechanical holder.

FIG. 3 is an exemplary system for delivering a sample solution to achip-based electrospray device for forming an electrospray plume tointroduce a sample into an orifice of a mass spectrometer.

FIG. 4 shows a schematic representation a mass spectrometer.

Like reference symbols in the various drawings indicate like elements.

DESCRIPTION

Dried blood spots (DBS) or dried matrix spots (DXS) where X can be anysample type on an sample card such as paper or other substrate surfacessuch as dried blood spots (DBS) on a paper or other substrate surfacesare viewed as an alternative to the conventional methods of collecting,transporting, and storing biological samples such as urine, plasma,saliva and feces as well as milk and other samples of interest. Amechanical holder that provides for a confined sampling region forextraction and removal of chemical substances contained in/on thesurface. The extracted substances can be analyzed using a massspectrometer.

FIGS. 1A-1C show a mechanical holder system 10 that provides a confinedsampling region for extraction and removal of chemical substances orsamples collected and stored on a variety of disposable, single usecollection substrates such as a dried matrix spot included on asubstrate such as a substrate formed of paper or similar absorbent ornon-absorbent substrate material. The mechanical holder system 10 formsa region in which a micro liquid junction between a solvent deliverydevice (e.g., a pipette tip or extraction tip that includes SPE sorbentmaterial) and a porous surface such as a cellulose or cotton-based cardor suitable polymeric membrane material and provides a method forsolvent extraction from the defined, constrained region. The mechanicalholder system enables analysis of a sample extracted from the collectionsubstrate, for example by a mass spectrometer. More particularly, FIG.1A shows the mechanical holder system 10 in an open position, FIG. 1Bshows the mechanical holder system 10 in an open position with acollection substrate placed in the holder system 10, and FIG. 1C showsthe mechanical holder system 10 in an closed position.

The mechanical holder system 10 includes a lower bed portion 16 and anupper component 12. The lower bed portion 16 and the upper component 12are mechanically connected by a hinge 34. In other embodiments, thelower bed portion 16 and the upper component 12 are mechanicallyseparated. In use, a collection substrate or sample card 50 such as adried matrix spot card or dried blood spot card is placed within thelower bed portion 16 such that the collection regions of the sample card50 are aligned with circular ridges 24 in the upper component 12 (e.g.,as shown in FIG. 1B). When the upper component 12 is closed or loweredon to the lower bed portion 16, the circular ridges 24 are clampeddirectly onto the sample substrate surface (e.g., the surface of samplecard 50) to define constrained surface extraction regions for eachsample. The constrained surface extraction regions formed by themechanical holder system 10 provide a substantially liquid tight sealaround a portion of the collection substrate (e.g., at least about 90%of liquid dispersed onto the substrate is collected, at least about 95%of liquid dispersed onto the substrate is collected, at least about 98%of liquid dispersed onto the substrate is collected). A sample can becollected from one of the constrained surface extraction regions bysequentially dispensing and aspirating an extraction/electrospraysolvent onto the sample surface in the extraction region using apipette. After collection, analytes extracted from the sample areanalyzed using mass spectrometry.

More particularly, the lower bed portion 16 is constructed to supportand contain a collection substrate/sample card 50, for example, a driedmatrix spot card or DBS card as shown in FIG. 1B. Exemplary collectionsubstrates can be made of various pure paper materials and/or polymericmaterials or other forms of membrane-like surfaces. In some examples,collection substrates such as matrix spot cards including blood spotcards include multiple sample locations each of which includes aseparate collection regions 52 such as dried sample spots for analysis.In some examples, the lower bed portion 16 can include outer rails 32configured to align the dried collection substrate with a cavity 14 inthe lower bed portion 16 and secure the sample card 50 in the cavity 14.In such examples, the cavity 14 can be configured to have a size andshape that is similar or the same as the size and shape of a collectionsubstrate. For example, dried matrix spot cards or similar surfacesupported analytical specimen cards have known widths ranging from 1 cmto 10 cm and known lengths ranging from 1 cm to 20 cm and the cavity 14can be configured to be approximately the same size as the size of thesample card to allow the sample card to be inserted into the cavity 14and secured therein. Providing a cavity 14 in the lower bed portion 16can provide the advantage of securing the collection substrate in aknown position within the mechanical holder system 10.

The lower bed portion 16 also includes a sample region 18. The sampleregion 18 is located within the cavity 14 in the lower bed portion 12 ata position that aligns with sample regions on the sample card 50. Thesample region 18 includes a raised surface (e.g., raised in relation tothe surface of other portions of the cavity 14) and includes multiplesample areas defined by ring-shaped depressions 20 in the sample region18. More particularly, both the sample region 18 and a middle portion 22of a particular sample area can be raised in relation to the surface ofother portions of the cavity 14. The ring shaped depressions 20 arealigned with cylindrical fittings 24 in the upper hinged component 12 asdescribed herein.

The upper component 12 provides a top surface that, when closed,encloses the sample card 50 within the mechanical holder system 10. Theupper component 12 is connected to the lower bed portion 16 by a hinge.The upper component 12 can also include one or more locking mechanisms,e.g., locking mechanisms 29 a, 29 b, 29 c, and 29 d configured to securethe upper component 12 to the lower bed portion 16 when the uppercomponent 12 is closed. The upper component 12 is configured to provideseparate regions for analysis of each of the dried matrix spots 52 onthe sample card 50. For example, as shown in FIGS. 1A-1C, the uppercomponent 12 includes four (4) cylindrical fittings 24 each having arigid perimeter surface 26 on the bottom which press down tightly on thedried matrix spot paper or substrate material of sample card 50 when theupper component 12 is lowered and clamped down to the lower bed portion16. The rigid perimeter surface 26 can have a width of between 0.1 mm to1 mm (e.g., 0.1 mm, 0.2 mm, 0.25 mm, 0.3 mm, 0.5 mm, 0.75 mm, or 1 mm).In some additional embodiments, the fittings 24 can be configured foranalysis of a larger regions of a sample and include a rigid perimetersurface 26 having a width of between 2 mm to 5 mm (e.g., between about 2mm and about 3 mm, between about 3 mm and about 4 mm, between about 4 mmand about 5 mm). In one exemplary embodiment, these fittings 24 arecompression fittings made of PEEK (poly ether ether ketone), polymer,brass or stainless steel which are commercially available from UpchurchScientific, Inc. or other suitable vendors. In some embodiments, thefittings 24 are made of a rigid plastic. The fittings 24 are held inplace in the top portion of the described device by threaded holes (see,e.g., FIGS. 2A and 2B). The lower portion of the fittings 24 (e.g., theportion which extends from the lower surface of the upper component 12to contact the sample card 50) are the shape of a compression fittingferrule or similar configuration and provide a rigid, sharp and circularperimeter surface which may be pressed firmly onto, for example, a papersurface.

The fittings 24 include an opening sized to provide an adequate sampleregion and to receive a solvent delivery device (e.g., a pipette tip orextraction tip) for sample extraction. For example, the fittings caninclude an opening that is 1/16 inch in diameter for smaller sample sizeapplications of approximately 1 mm in diameter or and/or ⅛ inch fittingswhich provide a sample extraction section of approximately 2-5 mm. Otherfitting sizes are can also be used. Each of these fittings may becentered on the dried matrix spot region or sample application region onthe paper or other substrate region to be analyzed.

As shown in FIG. 1C, the fittings 24 include an upper, open portion 40configured to receive a solvent delivery device such as a pipette tip oran extraction tip that includes SPE sorbent or packing material. Theupper, open portion 40 extends from the upper surface of the uppercomponent 12 (e.g., the surface which is not in contact the sample card50). For example, a pipette with an outside diameter of approximately 1mm and an inside diameter of approximately one-half millimeter can entera fitting opening 40 which is approximately 3 mm in diameter. Althoughthe larger end of the solvent delivery device is approximately 4 mm,only the lower one-third of the solvent delivery device enters into thePEEK ferrule so there is ample space to accommodate the solvent deliverydevice within the PEEK ferrule fitting. In an automated system, the X, Ypositioning (100 microns+/−50 microns) of the TriVersa NanoMate robot orits equivalent has sufficient precision and accuracy to reproduciblyplace the solvent delivery device (e.g., the pipette tip or extractiontip) into each fitting from sample to sample. The Z direction orcloseness of approach by the tip of the solvent delivery device to thesample surface is similarly controlled by the robot to 100 microns+/−50microns. Alternatively, one or more of the above actions can beperformed manually.

As noted above, the upper component 12 includes one or more lockingmechanisms, e.g., locking mechanisms 29 a, 29 b, 29 c, and 29 dconfigured to secure the upper component 12 to the lower bed portion 16when the upper component 12 is closed. In the example shown in FIGS.1A-1C, each of the locking mechanisms 29 a, 29 b, 29 c, and 29 dincludes a moveable arm attached to the lower bed portion 16 by screws28 a, 28 b, 28 c, and 28 d, respectively. The screws 28 a, 28 b, 28 c,and 28 d allow the locking mechanisms 29 a, 29 b, 29 c, and 29 d to berotated and positioned above the upper component 12. The end of the armportions each include a second screw 30 a, 30 b, 30 c, and 30 d to applypressure to the upper surface of the upper component 12 when tightened.To lock the upper component 12 to the lower bed portion 16, the screws30 a, 30 b, 30 c, and 30 d are tightened to press the rigid perimetersurface 26 of the fittings 24 on the upper surface into the ring shapeddepressions 20 in the sample region 18 on the lower bed portion 16. Aspacer can be included in the screw arrangements to ensure that screws30 a, 30 b, 30 c, and 30 d are tightened in a manner to providesubstantially uniform pressure onto the surface of the upper component12.

FIGS. 2A and 2B show a portion of the mechanical holder system 10 in aclosed arrangement with the locking mechanisms 29 a, 29 b, 29 c, and 29d disengaged and engaged, respectively. As shown in FIG. 2A, when theupper component 12 is lowered the rigid perimeter surface 26 of thecylindrical fittings 24 contacts a surface of the sample card 50. Asshown in FIG. 2B, when the locking mechanisms 29 a, 29 b, 29 c, and 29 dare engaged, the cylindrical fittings 24 are clamped down tightly anduniformly, such that the circular rigid perimeter surface 26 of thecylindrical fittings 24 extends into the ring shaped depressions 22 inthe sample region 18 and forms a liquid leak-tight seal between thesurface of the fitting 24 and the tightly compressed paper surface ofthe card 50 supported by the aluminum lower bed portion 16. In someexamples, the engagement of the locking mechanisms 29 a, 29 b, 29 c, and29 d depresses the card by between about 0.015 inch to about 0.030 inchto form the liquid leak-tight seal.

After placement of the sample card 50 in the mechanical holder system 10and engagement of the locking mechanisms 29 a, 29 b, 29 c, and 29 d, arobotic arm picks up a solvent delivery device (e.g., a conductivepipette tip or an extraction tip) and moves the solvent delivery deviceto the extraction solvent reservoir in order to pick up a 1-10microliter aliquot of an extraction/spray solvent. In some additionalembodiments, the fittings 24 can be configured for analysis of largerregions of a sample and 10-25 microliter aliquot of an extraction/spraysolvent can be used. Then, the solvent delivery device is positionedwithin the fitting 24 above the surface of card 50 to form a microliquid droplet or solvent pool maintained between the solvent deliverydevice and the sample surface. A sequence of repetitivedispense-aspirate steps affects an extraction of the sample surface.Then, the solvent delivery device is moved by the robot to the inlet ofthe microfabricated ESI chip and infusion nanoESI mass spectralacquisition is achieved employing either positive or negative iondetection. Exemplary solvent delivery devices are described, forexample, in U.S. Ser. No. 12/960,037 filed Dec. 3, 2010, the contents ofwhich is hereby incorporated by reference in its entirety

Thus, an opening of the center portion of the fitting 24 confines thesolvent extraction region to a defined area that is equal in size to theopening of the fitting. This allows a small volume of extraction solvent(e.g., 70% methanol, 30% water, 0.1% formic acid) to be dispensed intothe fitting 24 and hence onto the surface of the sample card 50. Thedispensed liquid forms a micro liquid droplet or small reservoir or poolof liquid solvent between the solvent delivery device and the samplesurface such as a dried matrix spot (DXS). The osmotic dispersion of thesolvent 60 can only spread to the inner wall surface of the fitting 24which is clamped and pressed tightly to the sample surface. Thisrestricted dispersion of the solvent limits the total volume of solventrequired to extract chemicals from the DMS and hence maintains a higherconcentration of analytes in the extract. The latter is beneficial forconcentration-sensitive detectors such as electrospray massspectrometry. In the absence of this confined region the extractionsolvent could disperse to the extremities of the sample card andsubstantially dilute the sample and/or limit the quantity of materialthat may be aspirated back into the solvent delivery device foranalysis. This can occur when the surface is hydrophyllic or highlyabsorptive of the solvent as is often the case in such applications.

The sample card holder forms reservoirs in the fittings 24 that have aminimum diameter of between about 0.5 to about 3 mm and the distancebetween the solvent delivery device (e.g., the pipette tip or extractiontip) and the sample surface is minimized to between about 0.5 to about 3mm. In some additional embodiments, the fittings 24 can be configuredfor analysis of larger regions of a sample and have a minimum diameterof between about 3 mm to about 5 mm. Thus, in the example of fittingshaving a minimum diameter of between about 0.5 to about 3 mm the volumeof solvent would range from about 0.98 cubic mm to about 21.2 cubic mm(volume=3.14*r²*height). A portion of the respective total volumedispensed (a residual) will remain on the porous sample surface (cardthickness might range from 0.01 mm to 0.5 mm), but the majority of theextract solution would be aspirated into the pipette tip or extractiontip for subsequent electrospray mass spectrometric analysis. Forexample, greater than 90% (e.g., greater than 90%, greater than 93%,greater than 95%, greater than 97%, greater than 98%) of the volume ofdispensed solution would be aspirated into the pipette tip or extractiontip.

Alternatively or additionally, one can treat the sample surface withhydrophobic chemicals such as Rain X or similar products which arefluorocarbon or silicone sprays with water repellent properties forcarpets, fabrics, etc. such that the solvent does not dispersethroughout the paper or other absorbent surface. The described devicewith its confined rigid perimeter surface limits the unwanted dispersionof solvents through the sample surface substrate and precludes requiringadditional treatment of the sample prior to analysis.

While the above description provides an example of a sample card holderwith four fittings, other configurations are possible. For example, thesample card holder can include multiple rows of fittings forming amatrix of analysis locations. The number of fittings can vary based onthe number of dried matrix spots or other analysis locations on thesurface of the sample card to be analyzed. For example, the sample cardholder can have fewer (e.g., one, two, three) fittings or a greaternumber of fittings (e.g., five, six, seven, eight, nine, ten, between10-20, between 20-30, or greater than thirty fittings). In someadditional examples, the sample card holder can include an 8×12 array offittings. In some additional examples, the sample card holder caninclude 96 fittings to align with a 96-well plate arrangement. In someadditional examples, the sample card holder can be configured to receiveand house multiple sample cards such as dried matrix spot cards or driedblood spot cards. In such examples, the number of fittings on the uppersurface is associated with the total number of analysis locations on themultiple dried matrix spot cards.

In some examples, the described sample card holder can be employed in arobotic solvent delivery and analysis system such as a robot similar tothe NanoMate, TriVersa NanoMate (Advion BioSystems, Ithaca, N.Y. USA),or related automated device equipped with a mandrel which is capable ofpicking up a pipette tip or other disposable solvent delivery devicesuch as an extraction tip. In one embodiment of the contemplated devicea robotic device picks up a clean solvent delivery device, aliquots asmall volume of extraction solvent (1-50 microliters) from a nearbysolvent reservoir and then dispenses a portion of the extraction solventwithin the sample card holder fitting such that a liquid junction isformed within the fitting between the sample surface and the solventdelivery device. This step affords an extraction of soluble compoundscontained within the DBS sample surface into the extraction solventcontained within the solvent delivery device. Next the robotic devicewithdraws the extract solution from the DBS surface and delivers thissolution containing dissolved chemicals/analytes to an analysis systemsuch as a mass spectrometer.

In a representative example a sample collected from a collectionsubstrate may be delivered to the inlet surface of a microfabricatedchip (FIG. 3) which houses a multitude of ESI emitters/electrospraynozzle sprayers which individually produce in a sequential manner anelectrospray plume directed to the inlet of an atmospheric pressureionization (API) mass spectrometer. This system may produce massspectral data which can be used for the qualitative or quantitativedetermination of chemicals/analytes of a wide diversity of types whichmay have been extracted from the sample card surface or similarsubstrate.

More particularly, FIG. 3 shows an exemplary mass spectrometry system100 used to identify the chemical composition of a compound based on themass-to-charge ratio of charged particles derived from the compound. Themass spectrometry system 100 includes a solvent delivery system 110 thatprovides appropriate elution solvents, usually of high organic solventcontent, to the extraction tip 300 (e.g., an extraction tip such as thetip described in U.S. Ser. No. 12/960,037 filed Dec. 3, 2010, thecontents of which is hereby incorporated by reference in its entirety).In other embodiments, a pipette tip that does not include SPE sorbent orpacking material can be used. While the example described in relation toFIG. 3 describes a solvent delivery system 110 capable of providingsolvent and applying a vacuum, in some additional examples, a syringecan be used to provide the solvent to the extraction tip or pipette tipand to aspirate the solvent into the extraction tip or pipette tip. Amandrel 120 is used to connect the extraction tip 300 to the solventdelivery system 110. The solvent delivery system 110 may also include adevice to provide a partial vacuum to the extraction tip 300 via aconnection through the mandrel 120. In some embodiments, the slightvacuum can be applied using a syringe. The solvent delivery system 110can be, for example, a liquid chromatograph such as a WatersnanoACQUITY, an Eksigent micro High Performance Liquid Chromatography(HPLC) system, or a Shimadzu liquid chromatograph equipped with a splitflow arrangement with a reduced flow of mobile phase directed to arobot. The robot may be a system such as the NanoMate™ or TriVersaNanoMate™ (Advion BioSystems, Ithaca, N.Y. USA) equipped with themandrel 120, which is capable of picking up a pipette tip or extractiontip from a multi-tip rack (e.g., 96 well plate, 384 well plate, 1536well plate.). In some embodiments, the mandrel is a tapered metal tubehaving an inner diameter of, for example, from about 1 mm to about 5 mm.(e.g., about 2 mm, about 4 mm, about 6 mm, about 10 mm).

During use, the robot may deliver the extraction tip 300 that includesan SPE packing material to the openings in the mechanical holder system10 to collect a sample from the surface of a sample card 50 (FIGS. 2Aand 2B). In other embodiments, a pipette tip that does not include SPEpacking material can be used. After collection of the sample, the robotmay deliver the extraction tip 300 to the inlet surface of amicrofabricated ESI chip 160 which houses a multitude of ESIemitters/electrospray nozzle sprayers 161. The end of the extraction tip300 is aligned and positioned at an opening of the nozzle sprayers 161.Thus, during use a solvent is provided from the system 110 through themandrel 120 and through the end of the extraction tip. As the solventpasses through the SPE packing material, the analytes retained thereinare dissolved in the solvent. Fluidic or hydrostatic pressure from thesolvent delivery system advances the eluting solvent or mobile phase andthe dissolved analytes to the end of the extraction tip 300. The fluidicpressure dispenses the dissolved analytes from the SPE tip by directingthe eluting solvent and dissolved analytes through the spray emitter 161that has an applied voltage. As the eluting solvent and dissolvedanalytes are expelled from the nozzle sprayer 161, an electrospray(e.g., a mist of small, charged droplets that can range from sub-micronsize under nanoelectrospray conditions to about 1-10 μm across) isformed. The electrospray is typically generated at or near atmosphericpressure and provides highly charged droplets of the solution containinganalytes. For example, microfabricated ESI chip 160 may include a 20×20array of nozzle sprayer 161 (or an array of 5×5 nozzles or 7×7 nozzlesor other similar arrangements of spray emitters in a microfluidic chiparrangement) having openings of between 2-50 μm in diameter (e.g., 2 μm,10 μm, 30 μm or 50 μm in diameter). Other types of sprayers including aspray probe or tube devices and a microfabricated sprayer device canalso be used. For example, a pipette tip that does not include SPEpacking material can be used. In other examples, rather than providing asolvent through the tip to force the analytes to be expelled through thesprayer 161, a slight Nitrogen gas pressure can be applied to the tipand to the several microliters of extract within the tip whichpushes/infuses the solvent extract through the ESI chip 160 foranalysis.

Upon passage through the nozzle sprayer 161 the electrospray is directedtowards the inlet orifice of an atmospheric pressure ionization (API)mass spectrometer 170. FIG. 4 shows a schematic representation of thecomponents within mass spectrometer 170. The electrospray droplets enterinto an atmospheric pressure interface (API), such as a capillary 218(while this example describes a heated capillary inlet, other inlets canbe used), that directs the ions from the electrospray into a vacuumportion 236 of the mass spectrometry system 170. As the droplets fromthe electrospray travel through the capillary 218, desolvation occurssuch that ions emerge from an exit 220 of the capillary 218. The ionsare directed through a skimmer 222 and the ions that emerge from theskimmer 222 are focused by a set of lenses 224 and into an ion opticsregion which may be a multipole region 228 or related or other type oflens focusing system. This multipole or ion optics region is typicallyoperated in the Rf-only mode and may be composed of a quadrupole,hexapole, octapole or similar ion optics device. In embodiments in whicha hexapole device is used as the multipole region 228, the ions arefurther guided through a quadrupole analyzer 230 or other mass analyzercapable of resolving ions with a mass-to-charge ratio and into adetector 232. The detector 232 amplifies the weak ion current signal ofthe sample ions. The detector may include an electron multiplier, photomultiplier or other suitable detector.

The solvent delivery from the solvent delivery device may range from aslow as 20 nL/min or as high as 50 microliters/min. The analyte(s) elutedfrom the solvent delivery device may be ionized by an electrosprayprocess or other available atmospheric pressure ionization techniquessuch as atmospheric pressure chemical ionization (APCI) and detected bymass spectrometry to produce either a full-scan electrospray massspectrum in either the positive ion mode or the negative ion modedepending upon the chemistry of the eluted analyte(s) from the solventdelivery device. Alternatively, the detected analyte(s) may be detectedand analyzed by other common mass spectrometric acquisition modes suchas full-scan mass spectral acquisition, full-scan MS/MS acquisition,neutral loss scans, precursor ion scans, selected reaction monitoring(SRM), multiple reaction monitoring (MRM), selected ion monitoring (SIM)or other common, modern acquisition modes of mass spectrometry.Applications that may benefit from this approach include pharmaceuticaldrug discovery and development, clinical diagnostics to monitor markersof disease, Vitamin D, its analogs and other steroids, amino acids,small drug compounds and their metabolites, antibiotics, peptides andproteins, inborn markers of metabolism such as carnitines, vitamins,forensic compounds, chemical warfare agents, nutritional supplements,pesticides and other potentially toxic chemicals on food surfaces, etc.

In some embodiments, the system can be fully automated. In a fullyautomated system, the system would introduce the sample card or samplesurface to be analyzed followed by robotic analysis as described aboveand finally an automated procedure for removing the sample cardpost-analysis to make way for the next sample card. In some additionalembodiments, the system may be operated in a manual mode in which a usermanually performs one or more the actions described herein.

While in some of the examples above a micro liquid junction is formedbetween the solvent delivery device and the sample card, in someembodiments a micro liquid junction is not formed and instead the liquiddisperses into the paper/substrate material like the wetting of asponge. In this case given sufficient liquid in the wetted surface thepipette tip still aspirates liquid from the wetted sponge-like surface.

While in some of the examples above the lower bed portion 16 and theupper component 12 are mechanically connected by a hinge 34, in otherembodiments, the lower bed portion 16 and the upper component 12 aremechanically separated and are used in a format similar to that of apress.

While at least some of the description above focuses on the analysis ofdried blood spots, dried spots of other samples of interest (e.g.,urine, plasma, saliva, feces, milk, food, fruits, vegetables, etc.) canbe analyzed using the systems and methods described herein.

1. A method of extracting analytes from a sample card, the methodcomprising: placing a sample card that includes a dried sample in alower portion of a device configured to house the sample card; loweringan upper portion of the device to the lower portion, the upper portionincluding one or more fittings with each fitting defining an opening inthe upper portion and including a rigid lower edge; engaging one or morelocking mechanisms to compress the sample card substantially uniformlybetween the rigid lower edge of the one or more fittings and the lowerportion to form a substantially liquid tight seal around a portion ofthe sample card; introducing a solvent into a solvent delivery device;dispensing the solvent to contact a surface of the sample card;aspirating the solvent and dissolved analytes into the solvent deliverydevice from the surface of the sample card; interfacing the solventdelivery device to an automation device to deliver the solvent deliverydevice to an ion sprayer; and directing ions from the ion sprayer to amass analyzer.
 2. The method of claim 1, wherein the sample cardcomprises a dried blood spot card.
 3. The method of claim 1, furthercomprising repeating the steps of dispensing and aspirating prior tointerfacing the solvent delivery device to an automation device todeliver the solvent delivery device to the ion sprayer.
 4. The method ofclaim 1, further comprising repeating the steps of dispensing andaspirating at the same location on the sample prior to interfacing thesolvent delivery device to an automation device to deliver the solventdelivery device to the ion sprayer.
 5. The method of claim 1, whereindispensing the solvent comprises dispensing microliters of solvent fromthe first end of the solvent delivery device to contact the sample. 6.The method of claim 1, wherein engaging one or more locking mechanismsdepresses the sample card by between about 0.015 inch to about 0.030inch to form the substantially liquid tight seal.
 7. The method of claim1, wherein engaging one or more locking mechanisms depresses the samplecard by between about 0.010 in to 0.100 to form the substantially liquidtight seal.
 8. The method of claim 1, wherein engaging one or morelocking mechanisms depresses the sample card by between about 0.025 inchto about 0.1 inch to form the substantially liquid tight seal.
 9. Themethod of claim 1, wherein engaging one or more locking mechanismscomprises tightening one or more screws.
 10. The method of claim 1,wherein dispensing the solvent comprises forming a liquid junctionbetween a sample surface of the sample card and the solvent deliverydevice.
 11. The method of claim 1, wherein dispensing the solventcomprises forming a liquid contact between a sample surface of thesample card and the solvent delivery device.
 12. The method of claim 1,wherein dispensing the solvent comprises wetting a surface of the samplecard.
 13. The method of claim 1, wherein engaging the one or morelocking mechanisms comprises engaging multiple locking mechanisms toapply a substantially uniform pressure onto sample substrate surface bythe upper portion.
 14. A system, comprising: a lower portion configuredto house a sample card that includes a dried sample; an upper portionincluding one or more fittings each defining an opening in the upperportion configured to receive a solvent delivery device and including arigid lower perimeter edge; and one or more locking mechanismsconfigured to compress the sample card between the rigid lower perimeteredge of the one or more fittings and the lower portion to form asubstantially liquid-tight seal around a portion of the sample card whenthe upper portion and the lower portion are in a closed position and thelocking mechanisms are engaged.
 15. The system of claim 14, wherein thesolvent delivery device is capable of delivering an extraction solventfor extracting chemicals from the sample substrate material.
 16. Thesystem of claim 14, wherein the solvent delivery device comprises apipette tip.
 17. The system of claim 14, wherein the solvent deliverydevice comprises an extraction tip.
 18. The system of claim 14, whereinthe fitting comprises PEEK, polymer, brass or stainless steel with arigid lower circular perimeter edge capable of making a leak-tight sealwhen clamped down upon the sample card without cutting a disk from thesample card.
 19. The system of claim 14, wherein the upper portionincludes at least four fittings in an array.
 20. The system of claim 14,further comprising a robotic device configured to extract analytes fromthe substrate to form a solution from a surface of the sample card usinga solvent delivery device which can then be withdrawn from the surfaceand delivered to a mass spectrometer.
 21. The system of claim 14 whereinthe fitting is configured such that extraction solvent introduced intothe fitting is substantially confined within the walls of the fitting.22. The system of claim 21, wherein confining the extraction solventwithin the walls of the fitting precludes the need to employ hydrophobicspray chemicals to preclude said dispersion.
 23. The system of claim 21,wherein the liquid tight seal is configured to confine at least 90% ofthe extraction solvent within the walls of the fitting.
 24. The systemof claim 21, wherein the liquid tight seal is configured to confine atleast 95% of the extraction solvent within the walls of the fitting. 25.The system of claim 21, wherein the liquid tight seal is configured toconfine at least 98% of the extraction solvent within the walls of thefitting.
 26. A system, comprising: a lower portion configured to housean sample card; an upper portion mechanically connected to the lowerportion, the upper portion including a fitting or an array of fittingsconfigured such that when the upper portion and the lower portion are ina closed position, a substantially liquid tight seal is formed around aportion of the sample card.
 27. The system of claim 26, wherein theliquid tight seal is configured to confine at least 95% of theextraction solvent within the walls of the fitting.